This project hypothesizes that the mucus clearance (MC) system, a fundamental airways defense mechanism, responds to noxious agents. Stresses at the airway lumen with autocrine feed-back mechanisms that principally involve the apical surfaces of airway epithelia. These autocrine feedback systems reflect, in part, the regulated release of a spectrum of 5' nucleotides(purines and pyrimidines) across the apical membrane surfaces in response to lumenal stress. We speculate that purinoceptors (P2Y-Rs) in the apical membranes of airway epithelial cells mediate cellular responses to released nucleotides that are integrated into complex cellular responses that culminate in increased MC. We propose three Aims to test these hypotheses. Specific Aim 1 proposes to measure the basal nucleotide concentrations on the surfaces of airway epithelial cells. The elements that control these levels, release rates, and extracellular nucleotides metabolic rates, will be identified and the consequences of basal nucleotide release on airway epithelial function tested. Specific Aim 2 will test the hypotheses that specific lumenal stimuli that are relevant to the physiology of airways defense, e.g., increased shear due to high air flows during cough, high transthoracic pressures, and vibration, release nucleotides in a regulated fashion. This Aim will then go on to test the hypothesis that nucleotide release in an autocrine fashion regulates the rates of transepithelial ion transport and examine how regulation of the Ca2+i-activated component of Cl- transport is confined to the apical membrane. Finally, Specific Aim 3 will test the concept that the physiology of nucleotide release and regulation of ion transport are relevant in vivo. In the first sub-Aim, basal release rates, the relationship to ion transport regulation, and importantly, the relationship of regulated nucleotide release to increases in mucus clearance will be tested in a large animal model. The second sub-Aim will test the concept that basal and stimulated release of nucleotides are a feature of human upper and flow airways, that they regulate ion transport rates, and that they are important components of the regulation of MC. The overall goal of the Project is to test the hypothesis that in human subjects, regulated nucleotide release at the lumen of airway epithelia is a function critical to regulation of MC, and hence lung defense, and that this mechanism may be a target for therapeutic interventions directed at clearing secretions from airway of patients with lung disease.